Minigene splicing assay

Total RNA was extracted from HEK293T and HeLa cells at 48 h post-transfection using TRIzol reagent (Takara, Japan). The extracted RNA was treated with DNase I (Thermo Scientific, United States) and reverse transcribed using the ImProm-II™ Reverse Transcription System according to the manufacturer’s instructions (Promega, United States). To detect alterations in splicing, minigene-specific cDNA was amplified using plasmid-specific primers (Table 2). The PCR products were separated by 12% agarose gel electrophoresis. To characterize the splicing patterns, the PCR products were subjected to Sanger sequencing.

The amplified plasmid-specific primers.